Abstract In addicts, the psychological distress associated with withdrawal can lead to resumption of drug taking (relapse) to ameliorate this state, at the expense of more beneficial goals. Indeed, an inability to tolerate the psychological distress experienced while in pursuit of a challenging goal (referred to as low distress tolerance, DT) is characteristic of substance users and is predictive of eventual relapse. In clinical models of DT, subjects engage in progressively difficult behavioral tasks that eventually become virtually impossible to complete correctly. Subjects are given the option to quit the task early, and DT is defined as the duration of time the subject spends on the very difficult task before quitting. Thus, DT measures the ability of the individual to persist in challenging goal-directed behavior in the face of increasing psychological distress, and has recently become a focal point of treatment strategies for substance use disorders. However, few studies have investigated the causal relationship between DT and relapse or its underlying neurocircuitry due to the difficulty in directly assessing these relationships in clinical populations. To address this, the Carelli lab recently developed a rodent model of DT and demonstrated that low DT in rats predicted high cocaine-seeking behavior. Critically, this only held true when DT was measured following extended abstinence from drug, a period of time shown to result in numerous neuroadaptations and increased drug seeking in rodent models. The overall objective of this application is to use our rodent model of DT to examine underlying neural circuits mediating DT, and their causal role in this behavior. Since the dorsolateral prefrontal cortex exhibits dampened activity in cocaine users during the DT task, proposed studies will investigate the role of its rodent homologue, the prelimbic cortex (PrL), in DT. Additionally, PrL projections to the nucleus accumbens core (NAc core) and PrL afferents from the insula (INS) are also of interest, given their role in craving, drug-seeking, negative affect, and task persistence. Thus, using our rodent model of DT, the present application will examine the PrL-NAc core (Aims 1 & 2) and INS-PrL (Aims 3 & 4) circuits during DT throughout various stages of cocaine self-administration and abstinence. In Aim 1, I will employ electrophysiological methods to examine how PrL or NAc core activity, or their connectivity, during DT is altered by a history of cocaine and predicts cocaine seeking and taking. In Aim 2, I will use optogenetic tools to investigate if activation of the PrL to NAc core pathway can reverse the deficits in DT induced by prolonged abstinence from cocaine self-administration. In Aim 3, I will use similar electrophysiology methods to examine how INS activity and/or INS-PrL connectivity during DT is altered by a history of cocaine and predicts cocaine seeking and taking. Lastly, in Aim 4, I will determine if optical activation of the INS to PrL pathway can reverse the deficits in DT induced by abstinence from cocaine self-administration. Collectively, these studies will provide insight into neural circuits underlying DT, a recent focus of promising treatment strategies for humans suffering from substance use disorders.